A Novel and Rapid RP-HPLC Quantitative Method for the estimation of Canagli lozin in Human Plasma

Ajitha A1, Sujatha K*2, Abbulu K3 1Department of Pharmaceutical Analysis, CMR college of Pharmacy, Kandlakoya (V), Medchal Road, Hyderabad 501 401, India (ORCID ID-0000-0003-0270-2842) 2Department of Pharmaceutical Chemistry, Sri Ramachandra Faculty of pharmacy, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), No.1 Ramachandra Nagar, Porur, Chennai 600 116 India. (ORCID ID-0000-0001-6130-5412) 3Department of Pharmaceutics, CMR college of Pharmacy, Kandlakoya (V), Medchal Road, Hyderabad 501 401,India. (ORCID ID-0000-0002-3932-4315)


INTRODUCTION
Diabetes mellitus affects 425 million people in the world, which accounts for this number rising to around 629 million diabetes globally by 2045 (El lien, 2019).

Reagents and Chemicals
Canagli lozin hemihydrate and Dapagli lozin were obtained as gift samples. HPLC grade Acetonitrile and water were purchased from the local vendor (Make: Merck, Mumbai, India). Potassium di-hydrogen phosphate (ACS grade) and ortho-phosphoric acid (ACS Grade) were procured from the local supplier (Make: Rankem, Hyderabad, India).

Instrumentation and Chromatographic Condition
Chromatographic analysis was performed on Waters Alliance HPLC 2695 model equipped with quaternary pumps, Photodiode array detector and auto sampler. Empower 2 Software is used thorough out the analysis. The HPLC column used was Phenomenex Luna C18 (2) (4.6 x 150 mm, 5m). The mobile phase consist of 0.01N potassium di-hydrogen phosphate in water (pH 3.5±0.04 adjusted with 10% ortho-phosphoric acid solution) and Acetonitrile in the ratio of (45:55) % v/v. Filtered the mobile phase through 0.45 Mobile phase low rate was set at 1.0 ml/min and column oven temperature was set at 30 • ±2 • C. The detector wavelength was set at UV 222 nm. The auto-sample cooler temperature was set at 5 • ±2 • C. Chromatographic run time was monitored for 15 min.        Preparation of internal standard Solution (5 µg/ml) Accurately weighed and transferred 12.5 mg of Dapagli lozin in to a 50 ml volumetric lask added 30 ml of diluent and sonicated for 5 minutes and

94.580
Overall SD 2.9787 Overall CV (%) 3.15 NOTE: 0.5ml (500 µl) of the above internal standard solution is to be mixed with spiking blank plasma and working stock dilutions of analyte to get 5 µg/ml Dapagli lozin (internal standard).
Extraction procedure Using a 1.0 ml micro pipette transferred 750 µL of human plasma and 500 µL of internal standard solution, 250 µL of Canagli lozin spiking solutions into a 15 ml graduated dry centrifuge tube and vortexed for 30 sec. Added 1 ml of Acetonitrile into the above centrifugation tube and mixed. Vortexed the mixture for 3 min. Centrifuged the mixture in the centrifuge tube at 4200 rpm for 12 min. The supernatant liquid after the centrifugation was been iltered using 0.45 µ membrane ilter.

RESULTS AND DISCUSSION
Chromatographic conditions development Method inalization was performed on basis of retention time, USP Tailing factor, USP Resolution and peak areas found for Canagli lozin and that of internal standard. The mobile phase pH was inalized on the basis to shorten the run time (15 min) and different trials were performed during method optimization. Peak retention time of Canagli lozin and Dapagli lozin were found to be about 8.7 min and 4.1 min respectively. USP plate count for Dapagli lozin peak and Canagli lozin peak are found to be 3100 and 6500 respectively (from Medium QC (MQC) solution). USP tailing for Dapagli lozin peak and Canagli lozin peak are found to be 1.05 and 1.03 respectively (MQC solution). USP resolution between Dapagli lozin peak and Canagli lozin peak was found to be 13.7 (MQC solution). The reported method is with shorter run time and more number of aliquots can be analyzed in less time. Typical chromatogram of MQC solution obtained by optimized chromatographic condition is presented in Figure 2.

Method Validation (Bio-Analytical)
The developed RP-HPLC method for the estimation of Canagli lozin was validated by following ICH Guidelines.
System Suitability-Evaluation Evaluation of System suitability parameters was performed by giving 6 replicate injections of MQC concentration. Evaluation of System suitability was performed on every day sequence as the irst experiment. Results of system suitability evaluations are presented in Table 1.

Selectivity/Speci icity
Speci icity of the proposed method is validated by analyzing blank samples of the biological matrix (6 batches of di-potassium ethylene diamine tetra acetic acid blank plasma and extracted blank plasma). No peak was observed in the retention time of Canagli lozin (analyte peak) and that of Dapagli lozin (internal standard). A typical chromatogram of plasma extracted blank and chromatogram of internal standard are shown in Figures 3 and 4. Carryover was studied by injecting the blank sample after the sample (MQC) and no impact of carryover was found.

Linearity
The method linearity was demonstrated over the range of 0.06 to 2.4 µg /ml for Canagli lozin. The coef icient of correlation (R 2 ) value was found to be 0.999 (Limit: not less than 0.99). The relationship adequately describes the response of the instrument with that of concentration of Canagli lozin. The slope and y-intercept of the calibration curve were 0.108 and 0.001 respectively. A specimen calibration curve obtained during the precision and accuracy study of validation is presented in Figure 5. The back calculated nominal concentrations obtained for calibration standard are presented in Table 2 along with the mean calculated accuracy value.

Precision and Accuracy
Within day and between day precision and accuracy were studied by determining 6 replicates of Lower Limit of Quantitation (LLOQC), Low (LQC), MQC and High (HQC). Accuracy and Precision method validation parameter were evaluated by 6 replicate analysis for Canagli lozin at 4 concentration levels, i.e., 0.06 µg/ml (LLOQC), 0.18 µg/ml (LQC), 1.2 µg/ml (MQC) and 1.9 µg/ml (HQC). Within-day and between day accuracy of plasma samples were studied and excellent mean % accuracy were obtained with range varied from 97.84 % to 99.81 % for within day and 97.22 % to 100.00 % for between day respectively. Precision of canagli lozin in plasma samples were studied and found to be 0.23 % to 5.54 % for within day and 0.14 % to 4.01 % for between days respectively (Tables 3, 4, 5, 6 and 7).

Recovery
Recovery studies are performed by measuring the peak areas found for Canagli lozin and that of internal standard, by comparing the results of extracted sample with corresponding extracts of blank spiked with Canagli lozin post-extraction. Accuracy values obtained for Canagli lozin at LQC, MQC and HQC were found to be 93.42 %, 97.96 % and 92.35% respectively (Table 8). Overall average accuracy of Dapagli lozin (Internal Standard) was found to be 98.07%.

Short term stock solution stability
During bench-top solution stability studies, 6 replicates of LQC & HQC samples (0.12 and 3.2 µg/ml) were analyzed after 9 hours at controlled room temperature on the laboratory bench (about 25 • C). The mean stability of solution was determined and found to be 99.72 % for LQC and 99.99 % for HQC respectively.

Long term Matrix sample Solution stability at -28±5 • C & -80±5 0 C for 60 Days
Long term matrix stock solution stability for the Canagli lozin was studied at a concentration of LQC-HQC level after a storage period of about 60 days at -28 • C & -80 • C in refrigerator. The mean solution stability of Canagli lozin was 99.63 %, 99.96 % & 100.14 % and 99.97 % (Tables 9 and 10).

CONCLUSIONS
The novel method validated as per ICH guidelines met with the preset acceptance criterias. This novel bio-analytical method can be used for pharmacokinetic studies in the clinical laboratories.
rapid LCeMS/MS assay method for the determination of canagli lozin in human plasma by solid phase extraction technique and its application to a pharmacokinetic study. Future Journal of Pharmaceutical Sciences,G.,Babu,K. R.,Annapurna,N.,Vekariya,N Nanoparticles of ZrPO4 for green catalytic applications.